Viruses encode multifunctional non-structural proteins in order to accommodate as much coding potential as possible into their relatively small genomes. Many of these proteins often interact with multiple host cell factors to manipulate the host environment to make it more conducive to virus replication and to help the virus evade the immune system, for example. Various viruses also express non-coding RNA molecules such as microRNAs (miRNAs), but the roles of these miRNAs are much less well understood. It stands to reason that, as is the case for non- structural proteins, viruses would evolve to maximize the utility of the miRNAs. BK polyomavirus (BKPyV) is a small DNA virus that is ubiquitous in the human population. In healthy individuals, it persists in the urinary tract without causing obvious disease, but in transplant patients, uncontrolled virus replication has serious outcomes. BKPyV encodes two miRNAs from a single pre-miRNA precursor. These miRNAs downregulate expression of the viral tumor (T) antigens, multifunctional proteins that facilitate virus replication by stimulating S phase entry and recruiting the host DNA synthetic machinery to the viral chromosome. These miRNAs do not affect replication of disease-associated genetic BKPyV variants, but severely limit replication of the archetype, or wild type, virus that circulates through the population and establishes a persistent infection. In this proposal, we will test the hypothesis that like their protein counterparts, the miRNAs are multifunctional macromolecules that also target host factors to facilitate virus infection. The two aims are to identify host targets and begin to assess the role of those targets in BKPyV infection.